SPERM GLYCINE RECEPTORS AND ZONA-INITIATED EXOCYTOSIS

描述（由申请人提供）：哺乳动物顶体反应（AR）是受精过程中必不可少的精子头部胞吐事件。哺乳动物精子中存在一个神经元甘氨酸受体/氯离子通道（GlyR），它是卵细胞透明带（ZP）蛋白ZP3启动AR所必需的。这项资助的总体目标是增加我们对GlyR在zp启动的AR中的作用以及控制其激活所涉及的机制的理解。我们假设ZP直接或通过受体串扰激活GlyR，通过GlyR的Cl-外排至少部分负责导致AR必需的ca2 +内流的去极化，并且GlyR的激活涉及pka介导的磷酸化。在体外AR实验中，我们将分别研究小鼠ZP和人重组人ZP3 （rhZP3）对小鼠精子和人精子的影响。具体目标如下：测定人类和小鼠精子GlyR是否介导细胞内Ca2+ (Ca2+i)在AR期间由rhZP3、小鼠ZP或甘氨酸引起的增加。测定人和小鼠精子GlyR是否参与甘氨酸、rhZP3或小鼠ZP引起的膜电位变化。3. 确定GlyR蛋白激酶A （PKA）磷酸化是否参与甘氨酸、小鼠ZP或rhZP3引发的AR。4. 确定PKA是否对GlyR介导的膜电位变化和甘氨酸、小鼠ZP和rhZP3诱导的Ca2+i增加有重要作用。5. 确定小鼠zp介导的膜电位和Ca2+i变化是否会在GlyR突变小鼠的精子中缺失或减少。6. 确定GlyR突变小鼠精子的AR是否发生在体外完整小鼠卵子的ZP上。如果没有，则确定在服用ZP时，这些精子中的黄体酮是否会刺激AR和受精？将人类精子和rhZP3的结果与小鼠精子和小鼠体内合成的ZP的结果进行比较，将有助于我们确定哺乳动物物种中是否存在共同的机制。

DESCRIPTION (provided by applicant): The mammalian acrosome reaction (AR) is a sperm head exocytotic event essential to fertilization. A neuronal glycine receptor/Cl channel (GlyR) is present in mammalian sperm and is necessary for the AR initiated by the egg zona pellucida (ZP) protein ZP3. The overall goal of this grant is to increase our understanding of the role of the GlyR in the ZP-initiated AR and the mechanisms involved in controlling its activation. We hypothesize that ZP activates the GlyR directly or via receptor cross-talk, that Cl- efflux through the GlyR is at least partly responsible for depolarization leading to Ca 2+ influx essential to the AR and that activation of the GlyR involves PKA-mediated phosphorylation. The effects of mouse ZP and human recombinant human ZP3 (rhZP3) on mouse sperm and human sperm respectively will be studied using imaging, photometry and antagonists in in vitro AR assays. The following are the Specific Aims. 1. Determination of whether the human and mouse sperm GlyR mediate the intracellular Ca 2+ (Ca2+i) increase elicited by rhZP3, mouse ZP or glycine during the AR. 2. Determination of whether the human and mouse sperm GlyR are involved in the membrane potential change elicited by glycine, rhZP3 or mouse ZP. 3. Determination of whether phosphorylation of the GlyR by protein kinase A (PKA) is involved in AR initiated by glycine, mouse ZP or rhZP3. 4. Determination of whether PKA is important to GlyR mediation of membrane potential change and to the Ca2+i increase elicited by glycine, mouse ZP and rhZP3. 5. Determination of whether mouse ZP-mediated membrane potential and Ca2+i changes would be absent or decreased in sperm from mice with GlyR mutations. 6. Determination of whether the AR of sperm from mice with GlyR mutations occur on the ZP of intact mouse eggs in vitro. If not, determination of whether the AR and fertilization be stimulated by progesterone in these sperm while they are on the ZP? Being able to compare results obtained with human sperm and rhZP3 and those obtained with mouse sperm and mouse ZP synthesized in vivo will help us determine whether common mechanisms are involved in mammalian species.